Downregulation of Rab23 inhibits hepatocellular carcinoma by repressing SHH signaling pathway

Abstract Hepatocellular carcinoma (HCC) is the sixth most common malignant tumors and the third leading cause of cancer‐related death worldwide. As an oncogene, Rab23 has been shown to be significantly related to the growth and migration of hepatocellular carcinoma in both in vitro and in vivo studies, but its underlying mechanism remains obscure. In the present study, we examined the effect of inhibiting Rab23 expression on the pathological progression of HCC. The correlation between liver Rab23 gene expression and survival probability in human HCC patients was analyzed using the TCGA database and CPTAC database. Rab23 knockdown hepatocellular carcinoma cell line was generated through lentiviral transduction, then we established a nude HCC xenograft model by subcutaneously implanting the transfected cells. The analysis of gene and protein expression was carried out using Western blot or RT‐qPCR, respectively. Flow cytometry analysis was used to detect the level of apoptosis. The expression levels of key proteins involved in the Sonic Hedgehog (SHH) signaling pathway were assessed. The results showed that HCC patients with low levels of hepatic Rab23 mRNA and protein had a better survival tendency than those with higher levels of Rab23. Cell proliferations were reduced and apoptosis levels were increased after Knocking down Rab23 in HCC cell lines. Furthermore, in vivo studies have demonstrated that suppression of the Rab23 gene results in decreased tumor size, proliferation rate, and reduced levels of SHH‐related proteins Smoothened and GLI‐1. The above results suggest that Rab23 is involved in the pathological progression of HCC as an important regulator of the SHH signaling pathway, which also provides an important research basis for new therapeutic strategies for HCC.


| BACKGROUND
The incidence of hepatocellular carcinoma (HCC) has increased worldwide, not only in East Asia but also in western Europe and the United States.HCC currently ranked sixth in incidence rate and third in mortality rate among all malignant tumors. 1 It is estimated by The World Health Organization that over 1 million patients will die from HCC by 2030. 2 Unfortunately, the current diagnosis rate of HCC is very low, and effective clinical treatments are lacking. 3HCC is a complex process involving multiple risk factors, including hepatitis B virus (HBV), Hepatitis C virus (HCV), cirrhosis, and alcohol use, which further promote abnormal activation of specific molecular signaling pathways, leading to disruption of the balance of oncogenes and tumor suppressor genes in cells, respectively. 4Currently, various oncogenes such as TP53, CTNNB1, AXIN1, and ARID1A have been identified to promote the initiation and progression of HCC by inducing abnormalities in cell proliferation, apoptosis, and signal transduction. 5,6In addition, providing a clear understanding of the relationship between these oncogenic genes and HCC, as well as comprehending their essential mechanisms in tumor progression, will contribute to the development of targeted treatments and preventive measures for HCC.
0][11][12][13][14] Furthermore, studies have demonstrated that Rab23 represses hedgehog (Hh) activities Via GLI-2 and promotes the proteolytic cleavage of GLI-3 into its cleaved repressor form.In addition, Rab23 also appeared to regulate Hh pathway activity through Smoothened. 15Both Rab23 gene and SHH pathway have also been implicated in tumor invasion and migration of HCC through their regulation of cell growth. 16,17However, the potential mechanisms of Rab23 in HCC are not yet clear.
In previous studies, we have demonstrated that Rab23 exists in both GDP-and GTP-bound forms.Disrupting this binding or reducing the level of Rab23 has a significant impact on the expression and nuclear localization of Gli1 in human liver cancer cell line Hep3B and HepG2, ultimately leading to suppression of cell growth.Therefore, we conducted this study to investigate the role and in vivo molecular mechanism of Rab23 silencing in HCC.

| Datasets collecting and preprocessing
The TCGA_LIHC cohort survival analysis for this study used Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancerpku.cn/)online analysis, with the data from The Cancer Genome Atlas (TCGA, https://portal.gdc.Cancer.gov/).At the same time, the protein data obtained in this study is downloaded from the The Clinical Proteomic Tumor Analysis Consortium (CPTAC, https://proteomics.cancer.gov/programs/) database.

| Survival analysis
First, HCC patients were divided into Rab23 high group and Rab23 low group according to the median Rab23 as a cut-off value.Load the R package "survival", then create the Surv object, and use Kaplan-Meier to build an overall survival (OS) analysis model to plot the survival distribution curve between the Rab23 high group and the Rab23 low group.

| Cell culture
Both the liver cancer cell lines Hep3B and HCCLM3 (iCell Bioscience Inc, Shanghai, China) were used in this study.Hep3B cells were grown in high-glucose DMEM (Corning, China) medium, while HCCLM3 cells were cultured in RPMI-1640 (Corning, China) medium.Both types of cells were cultured in a medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at a temperature of 37 C and 5% CO 2 .

| Lentivirus transfection
Three specific shRNAs (Table 1) were designed to target the Rab23 gene, and the lentivirus was purchased from BioSCI Res Company (China).Before transfection, approximately 4 Â 10 5 Hep3B and HCCLM3 cells were plated in a 6-well plated.The lentivirus particles were added to the cells at a multiplicity of infection (MOI) of 10 overnight.Afterward, the medium was replaced with 2 mL of fresh complete medium and incubated for an additional 48 or 72 h.Stable cell lines were obtained after 48 or 72 h of screening using 2 μg/mL puromycin (BeyoTime, China).Infection efficiency was calculated by observing GFP fluorescence under a fluorescence microscope.Quantitative real-time PCR or Western blotting was used to measure Rab23 expression in each stable cells line.

| Cell proliferation assay
The Cell Counting Kit-8 (CCK-8, Gongsheng, China) was used for cell viability analysis following the manufacturer's instructions.In detail, a total of 10 Â 10 3 cells/well of stable cells were plated onto a 96-well plate for 8 h and then treated with 10 μL of Cell Counting Kit-8 (CCK-8) solution (5 mg/mL) for 4 h.After that, the absorbance is measured at 450 nm in a microplate reader (FLx800, BioTek, Winooski, VT, USA).

| Flow cytometric analysis
The apoptosis level was evaluated using an Annexin V-APC Cell Apoptosis detection kit (BeyoTime, China) on the Guava EasyCyte flow

| Western blot assay
Total protein was isolated from cultured cells using lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% Glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM Sodium Pyrophosphate, 100 mM Sodium Fluoride) with a mixture of protease inhibitors including 1 mM PMSF and 10 μg/mL complete protease inhibitor mixture (Roche).The protein concentration was determined using a bicinchoninic acid protein assay kit (TaKaRa, Japan).The protein samples were run on 10% SDS-PAGE, transferred to PVDF membranes according to standard procedures.

| Correlation of hepatic Rab23 gene expression with survival probability in human liver cancer patients
As the role of Rab23 in HCC is not well-understood, we conducted an analysis to examine the correlation between hepatic Rab23 gene expression and survival probability in human HCC patients using the F I G U R E 3 Hep3B cells and HCCLM3 cells were treated with shRab23, viability assessment was detected by flow cytometer.Three technical repetitions for each analysis.
TCGA dataset and CPTAC dataset.Our analysis revealed that patients with lower levels of hepatic Rab23 mRNA and protein had better survival rates compared to those with higher levels of Rab23 (p < 0.05) (Figure 1A,B).Moreover, the expression level of Rab23 was also shown to be significantly and positively correlated with the stage of HCC (Figure 1C).

| Expression of Rab23 in liver cancer cell lines
We first examined the expression of Rab23 protein in wild and knockdown cell lines by Western blot analysis, respectively.The results showed that both Hep3B and HCCLM3 cells had similar levels of endogenous Rab23 protein expression (Figure 2A).We generated Rab23 knockdown cells in Hep3B cell lines using three different

| Rab23 knockdown suppresses GLI-1 expression
To evaluate the effect of Rab23 knockdown on SHH signaling pathway, the expressions of GLI-1 and cancer-associated fibroblast markers α-SMA in the shRab23 group were further detected.We found that when compared to WT control, both Rab23, GLI-1 and α-SMA protein levels were all significantly downregulated in shRab23 cells l ( p < 0.001 in Hep3B cells; p < 0.05 (GLI-1) or p < 0.01 (Rab23) in HCCLM3 cells) (Figure 4A-D).

| Proliferation inhibition of BALB/c xenograft tumor with shRab23 in vivo
To further validate the suppressive influence of Rab23 on tumorigenesis in vivo, we implanted Rab23 knockout HCCLM3 cells into nude mice and examined the enlargement of tumor cells.Compared with the shCrtl group, we found that tumors from shRab23 knockdown cell lines grew more slowly characterized by lower tumor tissue weight (p < 0.05) and smaller volume (Figure 5A-C).
H&E staining also indicated that tumor cells in the Rab23 knockout group had significantly reduced irregularly shaped tumor cell nucleosomes, incomplete cytoplasmic membranes, and heterogeneous chromatin structures compared to the control group (Figure 6D).However, according to photon flux detection, there were no significant differences of the metastasis of HCC between two groups (Figure 6A-C).
3.6 | Silencing of Rab23 suppress the progression of HCC in vivo via inhibiting the SHH signaling pathway Immunohistochemical (IHC) staining showed reduced Rab23 expression in knockout liver cancer cells (Figure 7A), along with decreased levels of KI-67 (Figure 7B), a proliferation marker for human tumors that has been strongly linked to advanced HCC. 18 Furthermore, the expression levels of the major SHH pathway proteins Smoothened (SMO) and GLI-1 were also significantly reduced

| DISCUSSION
HCC is a prevalent malignant tumor worldwide and is the primary cause of cancer-related deaths.In Asia such as China, 5-year survival rates are reported to be as low as 12%. 19Conventional treatments, including resection and anti-tumor medication, are not yet optimal due to the high diagnosis rate at advanced stages, leading to insignificant improvements in survival rates.
Rab23 is a small GTPase belonging to the Rab superfamily, whose gene is located on human chromosome 6p12.1. 8Over the past few years, it has been confirmed by several studies that Rab23 is an oncogene that plays a critical role in promoting cancer development. 101][22] Sicklick's study revealed Rab23 had elevated levels in human liver cancer tissues or liver cancer cells and was closely associated with tumor size. 23Zhang et al. 11 found that overexpression of Rab23 promoted the migration of Hep3B cells, and this process could be reversed by silencing Rab23.Currently, only a limited number of papers have reported on the role of Rab23 in HCC, and the specific molecular mechanisms by which Rab23 promotes the progression of liver cancer are far from being elucidated.
In previous study, we found that exhibited a positive expression rate of 53.5% in patients with HCC and was significantly associated with tumor size. 14The downregulation of Rab23 mRNA in Hep3B cells has also been shown to promote apoptosis and inhibit cell growth. 14Furthermore, we also demonstrated that the nuclear localization of Rab23 depends on its GDP/GTP binding state, and that the biological function of Rab23 is to control GTP hydrolysis as a molecular switch. 16In addition, Rab23 exhibited inhibitory effects on the transcriptional activity of Gli1 in HepG2 cells, although it was not directly regulated by the SHH signaling pathway. 16However, the

T
A B L E 1 shRNA sequences to target the Rab23 gene.Merck, USA).The cells were dissociated with 0.25% trypsin and washed twice with PBS after transfection.The cell pellets were resuspended in binding buffer and then mixed successively with 200 μL of 1Â apoptosis buffer.Annexin V-APC (5 μL) and PI (5 μL) were added, and the cells were stained for 15 min.Then, the cells were resuspended in 800 μL of 1Â apoptosis buffer and the final volume was adjusted to 1 mL.Next, 200 μL of the cell suspension was transferred to a 96-well plate (with three duplicate wells in each group) and subsequently analyzed using a flow cytometer.

2. 9 |
In vitro studies 2.9.1 | Human HCC xenograft model Eight-week-old BALB/c nude mice (GemPharmatech, China) were used for in vivo experiments and divided into two groups (shCtrl group and shRab23 group).The mouse had free access to standard feed and water in a temperature-controlled facility, which maintained a 12-hour light and 12-hour dark cycle.Tumorigenesis was induced by subcutaneous injection of mice with 4 Â 10 6 transfected HCCLM3 cells.The size of the tumor is regularly measured using a caliper at the same time point each week.Optical imaging was captured via the IVIS spectral imaging system (PerkinElmer, USA), and image analysis was conducted using the Living Image version 3.2 software.The nude mice were euthanized by injection of 3.5% aqueous solution of carbachol (0.1 mL/g) when the tumors F I G U R E 2 Expression levels of Rab23 and shRab23 in HCCLM3 cell lines and Hep3B cell lines, and cell viability analysis of these two cell lines.(A) The protein expression of Rab23 in HCCLM3 cell lines and Hep3B cell lines was detected by Western blot.(B) The protein expression of Rab23 in knockdown Hep3B cells was detected by Western blot.(C) The expression of Rab23 in lentiviral-transducted Hep3B cells was then measured by qRT-PCR.(D) Quantification of the expression of Rab23 protein in transfected Hep3B cells.(E) Western blot of Rab23 expression in knockdown HCCLM3 cells.(F) The relative mRNA expression of Rab23 was normalized to that of GAPDH in knockdown HCCLM3 cells.(G) Quantification of the expression of Rab23 protein in transfected HCCLM3 cells.Cell viability for infected (H) Hep3B and (I) HCCLM3 cell assayed by CCK-8.The data are presented as the mean ± SD.Significant differences between the shCtrl group and the shRab23 group: n = 3 per group, ***p < 0.001.reached an appropriate size.The tumor size is measured to further calculate the tumor volume using the formula length Â width Â 0.5.Each tumor tissue will be collected and weighed.All animal experiments were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

2. 10 |
Histological stainingCollected tumor tissues were paraffin embedded after fixed with 10% formalin fixation, and cut into appropriate thin slices.Hematoxylin and eosin (H&E) and Ki67 staining were performed according to a standardized procedure.Immunohistochemical analysis was used to detect the expression of Rab23 in tumor tissues using Rab23 antibody (Proteintech, USA).

2. 11 |
Statistical analysisDescriptive data were presented as means ± standard deviations (SD) s.The professional statistical analyses were performed using GraphPad Prism software version 5.01.Student t tests, one-way or two-way analysis of variance (ANOVA) were used to analyze the differences between the groups, respectively.A p value of <0.05 is considered statistically significant.

3 . 3 |
shRNA sequences targeting Rab23 (shRab23-1, shRab23-2 and shRab23-3).The gene expression of Rab23 significantly decreased in these knockdown cell lines compared to shCtrl ( p < 0.001), and the knockdown efficiency is a maximally of 70% (Figure2C).Moreover, all three shRNAs reduced the protein levels of Rab23 in cells with different knockdown efficiencies (22% for shRab23-1, 61% for shRab23-2, and 80% for shRab23-3) (Figure2B,D).We established a Rab23 knockdown cell line in HCCLM3 cells using the aforementioned shRNA sequences.Compared to the control group, both the gene level (Figure2F) and protein level (Figure2E,G) of Rab23 were significantly decreased.Among them, the knockdown efficiency of shRab23-1 was the highest, reaching nearly 90%.Rab23 knockdown decreases cell proliferation and induces cell apoptosis in liver cancer cellsWe next examined cell proliferation in these Rab23 knockdown cell lines.The cell proliferation levels of Hep3B cells at 72 h and HCCLM3 cells at 96 h were significantly lower than those of the shCtrl group (p < 0.001) (Figure2H,I).Furthermore, we investigated the impact of Rab23 knockdown on apoptosis levels in hepatocellular carcinoma cell lines using flow cytometry.The results showed that the apoptosis rate within both Hep3B and HCCLM3 cells was significantly increased in the shRab23 knockdown group compared to the shCtrl group (p < 0.001) (Figure3A,B).F I G U R E 4 Hep3B cells and were transfected by shRab23, protein expression of Rab23, GLI-1 and α-SMA were detected by Western blot (A).HCCLM3 cells were transfected by shRab23, protein expression of GLI-1 and Rab23 were detected by Western blot (B).The average expression levels of Rab23, GLI-1 and α-SMA in Hep3B cell lines were calculated by the ratio of Rab23, GLI-1 and α-SMA to GAPDH respectively (C).The average expression levels of Rab23, GLI-1 in HCCLM3 cell lines were calculated by the ratio of Rab23, GLI-1 to GAPDH respectively (D).Significant differences between the shCtrl group and the shRab23 group: n = 3 per group, *p < 0.05; **p < 0.01; ***p < 0.001.
in shRab23 liver cancer cells compared to the control (Figure 7C,D).Therefore, these findings indicated that Silencing of Rab23 may suppress the progression of HCC via inhibiting the SHH signaling pathway in vivo.F I G U R E 5 Silencing of Rab23 inhibited the tumor growth of hepatocellular carcinoma (HCC).Images of mice and tumors from the mice model and tumor size measurement (A).Images of tumor's weight (B).Images of tumor's growth curve (C).

F I G U R E 6
Representative images of fluorescence signals in nude mice detected by the International Veterinary Information Service system.The fluorescence signal intensity shows xenograft tumor size.(A) Tumor model of transplantation with control cells.(B) Tumor model of transplantation with shRab23 stably cells.(C) Measurement of fluorescence intensity.(D) H&E-stained liver sections exhibit the hepatic pathology with or without shRab23-1 of Rab23.above studies were all performed in vitro, and there is still a lack of evidence to support the function of Rab23 in vivo.Therefore, we conducted this present study to gain a deeper understanding of the molecular mechanisms underlying Rab23 by utilizing Xenograft mouse models.In our research, we generated a nude mouse xenograft model of HCC by subcutaneously implanting a HCCLM3 cell line with Rab23 knockdown.The findings demonstrated that Rab23 knockdown suppressed the growth of cancer cells and increased apoptosis in vivo.Importantly, we found that the deficiency of Rab23 resulted in a significant decrease in protein levels of both SMO and GLI-1.This suggests that Rab23 plays an essential role in liver cancer development and the SHH signaling pathway, which is consistent with our previous studies.The SHH pathway is a well-known signal transduction pathway comprising of main components such as hedgehog (Hh), the patched receptor(Ptc), a 12-domain transmembrane receptor, the Smoothened receptor(SMO), a 7-domain transmembrane receptor coupled to G protein-coupled receptor(GPCR), zinc finger transcription factor cubitusinterruptus (Ci), and other components that regulate embryonic development.24Numerous studies have indicated a close correlation between the aberrant activation of the SHH signaling pathway and the occurrence of various types of tumors such as cancers of skin, brain, liver, gallbladder, pancreas, stomach, colon, breast, lung, prostate, and hematological malignancies.[20][21][22]Sicklick et al. found the SHH signaling pathway plays an important role in the pathogenesis of HCC because of high expression levels of Ptc, SMO, and GLI-1 in liver cancer.23SMO is a major component involved in SHH signaling transduction, which can be coupled with G protein-coupled receptors (GPCRs) to inhibit the activity of suppressor of fused protein (SUFU) and GLI family of transcription factors.When the SHH signaling pathway is activated, PTCH-1, which destabilizes SMO and inhibits its activity, is degraded, leading to the activation of G-protein coupled receptor (GPCR)/SMO activity.25This promotes the dissociation of GLI from its inhibitor protein SUFU, resulting in the release and transport of GLI into the nucleus, triggering downstream target gene expression and activating cellular functions.26However, our results revealed a significant decrease in SMO and GLI-1 protein levels in tumor tissues, suggesting that Rab23 plays a negative regulatory role in the SHH signaling pathway and can suppress tumor growth in HCC.Overall, our study indicate that reducing Rab23 gene expression inhibit tumor cell growth and promoted apoptosis in vivo by downregulating the SHH signaling pathway.These experimental results present new evidence for the mechanisms of Rab23 on HCC, which will help to improve the development of therapeutic strategies for patients with hepatocellular carcinoma.AUTHOR CONTRIBUTIONS Yun-Jian Liu conceived and designed the present study.Si-Jia Liu analyzed and interpreted data.Yu-Wei Zang and Cui-Jun Huang performed experiments, wrote the manuscript.All authors approved the final version to be submitted.

F
I G U R E 7 The Sonic Hedgehog (SHH) signaling pathway were suppressed by Downregulation of Rab23.(A) The Rab23 protein expressions in liver cancer tissue with or without siRab23 were measured by immunohistochemical (IHC) staining.(B) The proliferation index Ki67 protein expressions in liver cancer tissue with or without siRab23 were detected by IHC staining.(C) GLI-1 and Smoothened protein expression in liver cancer tissue with or without siRab23 were checked by western blot; n = 5 per group.(D) The average expression levels of GLI-1 and Smoothened protein in liver cancer tissue were calculated by the ratio of GLI-1 and Smoothened to GAPDH respectively; n = 5 per group.